Wilkins-Port C E, McKeown-Longo P J
Cell and Molecular Biology Program and the Department of Physiology and Cell Biology (Mail Code 134), Neil Hellman Medical Research Building, Albany Medical College of Union University, 47 New Scotland Avenue, Albany, NY 12208, USA.
Biochim Biophys Acta. 1998 Sep 16;1404(3):353-66. doi: 10.1016/s0167-4889(98)00076-7.
The plasma protein vitronectin is thought to be an important regulator of extravascular plasminogen activation. In previous studies we have shown that a disulfide stabilized multimeric form of vitronectin is endocytosed and degraded by fibroblast cells (T.S. Panetti, P.J. McKeown-Longo, J. Biol. Chem. 268 (1993) 11988-11993; P.J. McKeown-Longo, T.S. Panetti, in: K.T. Preissner, S. Rosenblatt, C. Kost, J. Wegerhoff, D.F. Mosher (Eds.), Biology of Vitronectins and their Receptors, Elsevier Science Publishers, Amsterdam, 1993, pp. 111-118). The preparation of multimeric vitronectin used in these earlier studies was in the form of high molecular weight disulfide-bonded aggregates which were stable in sodium dodecyl sulfate (SDS). To address the question of whether vitronectin needed to be in the form of disulfide stabilized multimers in order to be endocytosed, a multimeric vitronectin, which was not disulfide stabilized, was prepared from vitronectin that had been treated with reducing agent and alkylated with iodoacetamide. The resulting protein migrated as a 65/75 kDa protein on SDS gels in the absence of reducing agent, confirming that this form of vitronectin was no longer stabilized into disulfide-bonded aggregates. However, the protein was still multimeric when analyzed by native gels and could be converted to SDS stable multimers by cross-linking agents. This result demonstrated that reduced and alkylated vitronectin aggregates into multimeric forms which are not stable in SDS. Similar to disulfide stabilized multimers, alkylated multimers of vitronectin bound to sulfated proteoglycans in the extracellular matrix and were endocytosed and degraded. Degradation of both forms of vitronectin was inhibited with arginine-glycine-aspartic acid peptides, an anti-alphavbeta5 antibody and heparin. Chloroquine and wortmannin were also able to inhibit degradation of both forms of vitronectin, indicating that both multimeric forms were following the same endocytic and degradative pathway. These results suggest that the organization of vitronectin into a multimeric form which will be recognized for endocytosis does not require disulfide bond stabilization. This study further suggests that recognition of vitronectin for endocytosis is dependent upon its conversion from a monomeric to a multivalent form (C.E. Wilkins-Port, P.J. McKeown-Longo, Mol. Biol. Cell 8:S:64A (1997).
血浆蛋白玻连蛋白被认为是血管外纤溶酶原激活的重要调节因子。在先前的研究中，我们已经表明，一种二硫键稳定的多聚体形式的玻连蛋白可被成纤维细胞内吞并降解（T.S. 帕内蒂，P.J. 麦基翁 - 隆戈，《生物化学杂志》268 (1993) 11988 - 11993；P.J. 麦基翁 - 隆戈，T.S. 帕内蒂，载于：K.T. 普赖斯纳，S. 罗森布拉特，C. 科斯特，J. 韦格霍夫，D.F. 莫舍（编），《玻连蛋白及其受体的生物学》，爱思唯尔科学出版社，阿姆斯特丹，1993，第111 - 118页）。这些早期研究中使用的多聚体玻连蛋白制剂是高分子量二硫键结合聚集体的形式，在十二烷基硫酸钠（SDS）中稳定。为了解决玻连蛋白是否需要以二硫键稳定的多聚体形式才能被内吞的问题，从用还原剂处理并用碘乙酰胺烷基化的玻连蛋白制备了一种非二硫键稳定的多聚体玻连蛋白。在没有还原剂的情况下，所得蛋白质在SDS凝胶上迁移为65/75 kDa的蛋白质，证实这种形式的玻连蛋白不再稳定形成二硫键结合的聚集体。然而，通过天然凝胶分析时该蛋白质仍然是多聚体，并且可以通过交联剂转化为SDS稳定的多聚体。该结果表明，还原和烷基化的玻连蛋白聚集成在SDS中不稳定的多聚体形式。与二硫键稳定的多聚体类似，玻连蛋白的烷基化多聚体与细胞外基质中的硫酸化蛋白聚糖结合，并被内吞和降解。两种形式的玻连蛋白的降解都被精氨酸 - 甘氨酸 - 天冬氨酸肽、抗αvβ5抗体和肝素抑制。氯喹和渥曼青霉素也能够抑制两种形式的玻连蛋白的降解，表明两种多聚体形式都遵循相同的内吞和降解途径。这些结果表明，玻连蛋白组织成可被识别用于内吞的多聚体形式不需要二硫键稳定。这项研究进一步表明，玻连蛋白被识别用于内吞取决于其从单体形式转变为多价形式（C.E. 威尔金斯 - 波特，P.J. 麦基翁 - 隆戈，《分子生物学细胞》8:S:64A (1997)）。
